Nucleocytoplasmic Shuttling of Influenza A Virus Proteins

Influenza viruses transcribe and replicate their genomes in the nuclei of infected host cells. The viral ribonucleoprotein (vRNP) complex of influenza virus is the essential genetic unit of the virus. The viral proteins play important roles in multiple processes, including virus structural maintenance, mediating nucleocytoplasmic shuttling of the vRNP complex, virus particle assembly, and budding. Nucleocytoplasmic shuttling of viral proteins occurs throughout the entire virus life cycle. This review mainly focuses on matrix protein (M1), nucleoprotein (NP), nonstructural protein (NS1), and nuclear export protein (NEP), summarizing the mechanisms of their nucleocytoplasmic shuttling and the regulation of virus replication through their phosphorylation to further understand the regulation of nucleocytoplasmic shuttling in host adaptation of the viruses.     

中国精神病人外周血博尔纳病病毒P24基因片段检测

目的 探讨博尔纳病病毒与人类精神疾病的关系。方法 采用套式逆转录酶聚合酶链反应(nested RT-PCR)结合荧光定量(FQ)PCR检测了31例精神分裂症病人,16例情感障碍病人以及50例健康人外周血单个核细胞(PBMC)中BDV P24基因片段。结果精神分裂症组BDV P24基因片段阳性率为9.7％(3/31);情感障碍组(0/16)和健康组(0/50)均为阴性。精神分裂症组阳性率高于健康组,但差异无显著性意义(P>0.05)。结论 中国的精神病人中存在BDV感染,但BDV感染是否与精神分裂症发病相关有待进一步研究。     

博尔纳病毒在宁夏的人和动物感染的检测

目的对临床VE患者以及其本人接触动物的检测,探讨BDV在宁夏地区的感染状况及感染机制,并分析BDV核酸和转化后的氨基酸序列,构建病毒种系发生的来源。方法应用巢式逆转录酶聚合酶链反应结合荧光定量PCR检测患者及其接触的动物的外周血单核细胞的p24和p40基因片段。对检测阳性标本进行连接测序,应用MEGA和DnaSP4.0软件对测序结果同国外的标准株HE80、H1766、strainV等进行核酸和氨基酸序列对比分析,并构建病毒基因的系统发生树。结果在60例VE患者及其接触的710绵羊中,PCR检测发现有3例阳性,阳性检出率5%;9头绵羊阳性,阳性检出率为1.27%。基因聚合分析显示人和动物BDV的核酸序列和编码氨基酸序列与德国马源性的HE80株同源性高达100%,重构基因系统发生树可见宁夏VE患者和牛羊BDV核苷酸序列与国外的标准株形成宁夏-德国-日本的混合支系,同时宁夏绵羊BDV序列也形成了一个独立的支系。结论宁夏地区人和牛羊存在BDV的自然感染,并且人的感染存在动物源性,其传染途径很可能是呼吸道的传播可能引起脑炎的非特异性的临床症状,病毒在种系发生中与德国马源性HE80株有极高的同源性,病毒可能由国外引入本土,不排除病毒株变异的可能,人和绵羊之间存在相互传染的可能。     

博尔纳病病毒核蛋白转染人少突胶质细胞的蛋白组学研究

目的 研究博尔纳病病毒(Borna disease virus)核蛋白(Nucleoprotein,p40)质粒转染人少突胶质细胞(Oligodendrocyte,OL)后蛋白表达变化。方法 构建BDVp40质粒转染OL细胞的细胞模型和空载体转染细胞模型,分别提取各组细胞蛋白进行双向凝胶电泳和图像分析,用HPLC-Chip/MS纳流液质联用技术进行差异蛋白分析,NCBI数据库搜索鉴定差异蛋白。结果 BDVp40转染后出现蛋白表达差异,与对照组相比有8个蛋白表达上调,4个蛋白表达下调,1个蛋白只在转染后细胞表达而未转染OL细胞组不表达,其中4个蛋白与神经系统疾病相关。结论 BDVp40转染OL细胞后导致相关蛋白表达改变,其中一部分蛋白与神经系统疾病相关。     

抗博尔纳病病毒磷蛋白单克隆抗体的制备及鉴定

目的以重组的博尔纳病病毒(BDV)磷蛋白(p24)为免疫原制备单克隆抗体,并鉴定其特异性。方法以纯化后的重组BDVp24免疫小鼠,将骨髓瘤细胞与其脾细胞进行融合,经ELISA法筛选出分泌抗p24单抗的细胞株,并对其进行克隆与亚克隆培养,将最终获得的其中2株细胞株分别注入小鼠腹腔制备单抗,经亲和纯化法纯化后,采用ELISA法测定效价,利用蛋白免疫印迹和免疫荧光鉴定其特异性。结果建立了2株稳定分泌抗p24单抗的杂交瘤细胞株,抗体亚类均为IgG1型。纯化后的腹水抗p24单抗纯度为98%和93%,ELISA效价在1∶81 000以上,该抗体均能识别重组p24蛋白和BDV感染人类少突胶质细胞(Oligodendroglia cell,OL细胞)所表达的p24蛋白。结论成功制备了灵敏性高、特异性好的抗BDVp24单抗,为博尔纳病病毒诊断方法的研制及感染机理的研讨提供依据。     

贵州遵义地区蝙蝠脑组织BDV-P24基因片段的检测

目的了解贵州遵义地区蝙蝠博尔纳病病毒(BDV)感染情况.方法 采用巢式逆转录聚合酶链反应(nRT-PCR)检测82例蝙蝠脑组织BDV-P24基因片段,阳性产物进行基因序列测定,同源性及分子系统树分析.结果 在82例蝙蝠脑组织中检测出7例BDV-RNA P24基因片段阳性,只有5例成功测出BDV-P24基因片段,与标准株马源Strain V相比较同源性高达99%,出现2个位点的一致性沉寂突变(nt1650T~C,nt1740G-T,突变率为0.9%),与H1766比较其同源性达97%,出现5个位点的一致性沉寂改变(nt1599A~G,nt1671T~C,nt1677T~C,nt1695G~A,nt1740G~T,突变率为2.2%),与He/80比较起同源性为96%,出现7个位点的一致性沉寂改变(nt1566 G~A,nt1581C~T,nt1659 T~C,nt1668 A~G,nt1674 T~C,nt1695 G~A,nt1740 G~A,突变率为3.1%).结论 贵州遵义绥阳地区蝙蝠存在BDV感染,与马源Strain V存在高度同源性,人感染BDV可能存在潜在的动物源性.     

牧羊犬外周血和脑组织博尔纳病病毒检测结果的比较

目的检测同一牧羊犬外周血和脑组织博尔纳病病毒(BDV)p24片段,比较两者阳性率的差异。方法采用改进的荧光定量套式RT-PCR,对新疆伊犁地区圈养的100只牧羊犬外周血单核细胞(PBMC)和脑组织(BT)同时进行BDV p24基因片段的检测,并对阳性标本采用GFP-p24、pMD-19扩增后电泳验证,排除质粒污染,并克隆测序,用Fisher精确检验和χ2检验分析两者阳性率的差异。结果牧羊犬外周血单核细胞和脑组织BDV p24检测的阳性率分别为5%和9%;PBMC组和BT组BDV p24检测阳性率差异无统计学意义(P>0.05)。结论BDV在脑内持续感染,但以RT-PCR对外周血单核细胞BDV p24片段的检测有可能替代脑组织BDV检测,作为大规模流行病学调查的手段。     

Pre-S Proteins in Chronic Hepatitis B Virus Infection: Markers of Active Viral Infection

The role of large (pre-S1) and middle (pre-S2) proteins of HBsAg in hepatitis B virus (HBV) infection is not fully known. Therefore, we studied the expression of pre-S proteins in the liver and serum of 26 patients with chronic HBV infection, using immunoperoxidase staining and enzyme immunoassay. Pre-S1 and pre-S2 proteins were detected in a large number of patients in both liver and serum, irrespective of the disease activity. Serial sections showed that most cells positive for HBsAg were also positive for pre-S proteins. The localization of pre-S2 and HBsAg was similar, with cytoplasmic and membranous stainings of hepatocytes, whereas pre-S1 was expressed exclusively in cytoplasm. Serum levels of HBsAg, pre-S1, and pre-S2 of DNA polymerase-positive cases were significantly higher than those of DNA polymerase-negative cases. Membranous display of pre-S2 on hepatocytes was observed more often in DNA polymerase-positive patients, and their serum pre-S2 levels were significantly higher than those without it. The predominant localization of cytoplasmic HBcAg usually was associated with active, ongoing hepatitis. Its expression and DNA polymerase activity were significantly correlated. These results indicate that pre-S proteins in serum and the membranous display of pre-S2 on hepatocytes of patients with chronic HBV infection refect active viral replication, but their expression does not correlate with disease activity.     

病毒性脑炎患者血液和脑脊液博尔纳病病毒p24核苷酸片段的检测

目的探讨博尔纳病病毒(Borna disease virus,BDV)与病毒性脑炎(Viral encephalitis,VE)的关系,以及贵州遵义地区是否存在BDV感染。方法采用巢式逆转录荧光定量PCR(FQ-nRT-PCR)检测了32例VE患者和34例对照组(非神经系统疾病外科手术患者)外周血单核细胞(peripheral blood monouclear cells,PBMC)和脑脊液(cerebrospinal fluid,CSF)中的BDV p24基因片段。结果VE患者PBMC和CSF中BDV p24基因片段阳性率均为12.5%。明显高于对照组(0%,Fisher精确概率检验:χ2=4.524,P<0.05);PCR扩增目的基因片段阳性产物测序后,与NCBI网站GenBank提供的BDV标准病毒株V和H1766病毒核苷酸序列比较同源性为95.35%和98.84%,在4个位点出现突变(nt1649T→C,nt1656G→A,nt1670C→T,nt1676C→T),突变率为4.65%;与H1766病毒株亲缘关系最近。结论遵义及其周边地区部分VE患者中存在BDV感染,并可能与病毒性脑炎发病有关。     

宁夏地区病毒性脑炎中新发博尔纳病病毒感染的研究

目的 研究宁夏地区新发博尔纳病病毒(BDV)在病毒性脑炎患者中的感染状况,分析病毒株的核酸序列、编码的氨基酸序列和病毒株的系统发生.方法 套式反转录实时荧光定量PCR检测60例病毒性脑炎患者血液标本;并对前期课题组检测的59例病毒性脑炎患者中BDV p24阳性样本和本实验PCR检测阳性标本使用ELISA法检测其对应脑脊液中p40抗体,对阳性标本进行连接测序,应用MEGA和DnaSP4.0软件对测序结果与国外标准株He/80、H1766、strain V等的核酸和氨基酸序列对比分析,构建病毒基因的系统发生树.结果 PCR检测119份血标本,发现有12份标本BDV p24阳性,阳性检出率为10.08%,7份BDV p40检测阳性,阳性率为5.88%;ELISA检测脑脊液核蛋白抗体,有2份阳性,阳性检出率为1.68%.基因聚合分析显示,7份BDV阳性的核酸序列中有6份与德国马源性的He/80株同源性达100%,1份仅有1个碱基发生同义突变;转录后的氨基酸对比分析显示,7份与He/80株完全一致;重构的基因系统发生树中可见宁夏病毒性脑炎患者BDV核苷酸序列与国外标准株形成一个中国(宁夏)-德国-日本的混合支系.结论 宁夏地区病毒性脑炎患者中可能存在BDV感染,其发生可能与动物的密切接触相关,病毒在种系发生中与德国马源性He/80株有极高的同源性,推断病毒有可能从国外传入本土,不排除病毒株变异的可能.     

贵州遵义地区山羊Borna病毒P24基因片段的检测

目的探讨贵州遵义及周边地区山羊博尔纳病病毒(BDV)感染状况。方法采用荧光定量套式逆转录酶聚合酶链反应(FQ-nRT-PCR)检测了300只山羊外周血单个核细胞(PBMC)中BDVP24基因片段。对阳性产物进行基因序列测定,氨基酸顺序,及同源性分析。结果300只山羊PBMC中2只检出BDVP24基因阳性片段。山羊BDVP24基因片段阳性率为0.67(。BDVP24基因片段测序结果与GenBank提供的strainV毒株比较同源性为96.51%,有3个位点出现一致性沉寂突变,与BDV/MDCK毒株比较同源性为96.51%,有3个位点出现一致性沉寂突变,与C6BV毒株比较同源性为96.51%,有3个位点出现一致性沉寂突变,但所编码的氨基酸没有改变。结论贵州遵义及周边部分地区山羊存在BDV自然感染。     

Impact of Nucleotide Mutations at the HNF3- and HNF4-Binding Sites in Enhancer 1 on Viral Replication in Patients with Chronic Hepatitis B Virus Infection

Background/Aims

The hepatitis B virus (HBV) genome contains binding sites for hepatocyte nuclear factors (HNF) 3 and 4 in the core domain of enhancer 1 (Enh1), and mutations in this domain have a strong impact on virus replication. We aimed to identify frequent base-mutation sites in the core domain of Enh1 and to examine the impact of these mutations on viral replication.

Methods

We studied virological characteristics and genetic sequences in 387 patients with chronic hepatitis B. We evaluated functional differences associated with specific mutations within the core domain of Enh1.

Results

Mutations in the core domain were found with significant frequency in C1126 (122/387 [31.5%], the binding site for HNF3) and in C1134 (106/387 [27.4%], the binding site for HNF4). A single mutation at nt 1126 (C1126) was identified in 17/123 (13.8%), and 105/123 (85.4%) had double mutations (C1126/1134). The level of HBV DNA (log10 copies/mL) was lower in single mutants (C1126, 5.81±1.25) than in wild (6.80±1.65) and double mutants (C1126/1134, 6.81±1.54). Similarly, the relative luciferase activity of C1126 and C1126/C1134 was 0.18 and 1.12 times that of the wild-type virus, respectively.

Conclusions

Mutations in the HNF3 binding site inhibit viral replication, whereas mutations at the HNF4 binding site restore viral replication.     

Peste Des Petits Ruminants Virus Infection of Small Ruminants: A Comprehensive Review

Peste des petits ruminants (PPR) is caused by a Morbillivirus that belongs to the family Paramyxoviridae. PPR is an acute, highly contagious and fatal disease primarily affecting goats and sheep, whereas cattle undergo sub-clinical infection. With morbidity and mortality rates that can be as high as 90%, PPR is classified as an OIE (Office International des Epizooties)-listed disease. Considering the importance of sheep and goats in the livelihood of the poor and marginal farmers in Africa and South Asia, PPR is an important concern for food security and poverty alleviation. PPR virus (PPRV) and rinderpest virus (RPV) are closely related Morbilliviruses. Rinderpest has been globally eradicated by mass vaccination. Though a live attenuated vaccine is available against PPR for immunoprophylaxis, due to its instability in subtropical climate (thermo-sensitivity), unavailability of required doses and insufficient coverage (herd immunity), the disease control program has not been a great success. Further, emerging evidence of poor cross neutralization between vaccine strain and PPRV strains currently circulating in the field has raised concerns about the protective efficacy of the existing PPR vaccines. This review summarizes the recent advancement in PPRV replication, its pathogenesis, immune response to vaccine and disease control. Attempts have also been made to highlight the current trends in understanding the host susceptibility and resistance to PPR.     

酒精性肝病合并乙型肝炎病毒感染60例临床分析

目的 通过对郑州大学第一附属医院5年收治的60例酒精性肝病(ALD)合并乙型肝炎病毒(HBV)感染患者的临床资料分析,研究其临床特点和危险因素,以提高大众对酒精性肝病合并乙型肝炎病毒感染的认识及增加对戒酒等健康生活习惯的重视.方法 分析60例酒精性肝病合并乙型肝炎病毒感染患者的临床资料,并以100例单纯酒精性肝病患者、100例单纯乙型肝炎病毒感染患者为对照组,比较组间疾病严重程度,评价3组的治疗效果,分析酒精性肝病合并乙型肝炎病毒感染发病类型的危险因素.结果 (1)合并组实验室指标升高明显高于对照组(P＜0.05).(2)治疗4周后合并组治疗效果较两对照组差(P＜0.05).(3)通过Logistic回归分析表明,危险因素OR值为平均每日饮酒量＞HB-DNA定量＞饮酒年限＞吸烟＞年龄.结论 乙型肝炎病毒感染可加重酒精性肝病患者病情,降低治疗效果;每日饮酒量、饮酒年限、HBV-DNA定量、吸烟、年龄均为二者合并时的危险因素;因此对于酒精性肝病合并乙型肝炎病毒感染患者通过戒酒,培养良好生活方式是预防肝功能损伤加重、改善疾病疗效及预后的重要积极措施.     

Autophagy Activated by Bluetongue Virus Infection Plays a Positive Role in Its Replication

Bluetongue virus (BTV) is an important pathogen of wild and domestic ruminants. Despite extensive study in recent decades, the interplay between BTV and host cells is not clearly understood. Autophagy as a cellular adaptive response plays a part in many viral infections. In our study, we found that BTV1 infection triggers the complete autophagic process in host cells, as demonstrated by the appearance of obvious double-membrane autophagosome-like vesicles, GFP-LC3 dots accumulation, the conversion of LC3-I to LC3-II and increased levels of autophagic flux in BSR cells (baby hamster kidney cell clones) and primary lamb lingual epithelial cells upon BTV1 infection. Moreover, the results of a UV-inactivated BTV1 infection assay suggested that the induction of autophagy was dependent on BTV1 replication. Therefore, we investigated the role of autophagy in BTV1 replication. The inhibition of autophagy by pharmacological inhibitors (3-MA, CQ) and RNA interference (siBeclin1) significantly decreased viral protein synthesis and virus yields. In contrast, treating BSR cells with rapamycin, an inducer of autophagy, promoted viral protein expression and the production of infectious BTV1. These findings lead us to conclude that autophagy is activated by BTV1 and contributes to its replication, and provide novel insights into BTV-host interactions.     

博尔纳病病毒p24和p40基因星形胶质细胞特异表达重组质粒的构建

目的 构建星形胶质细胞特异性表达的博尔纳病病毒磷蛋白和核蛋白基因重组质粒,以供后续实验研究使用。方法 用PCR方法扩增BDV p24和p40基因完整序列同时加入Nhe I和EcoR I酶切位点,将所得目的片段克隆至pMD18-T 载体,再通过酶切连接将其连接至pCMvie-GFAP质粒中的GFAP启动子的下游,分别得到p24和p40重组质粒。用酶切、PCR以及测序三种方法鉴定重组质粒,并将质粒转染人胶质瘤细胞株（U251）,采用免疫细胞化学方法检测目的蛋白表达。结果pCMvie-GFAP-BDVp24和pCMvie-GFAP-BDVp40重组质粒经酶切、PCR鉴定可见目的条带位置与预期相符,经测序可见插入序列正确,质粒转染U251细胞后,经免疫细胞化学检测到目的蛋白表达。结论 成功构建了pCMvie-GFAP-BDVp24和pCMvie-GFAP-BDVp40星形胶质细胞特异性表达质粒,为研究这两种病毒蛋白的致病机制及其对星形胶质细胞的影响提供了工具。     

Celiac Disease and Non-organ-specific Autoantibodies in Patients with Chronic Hepatitis C Virus Infection

OBJECTIVE: Considering that celiac disease (CD) is an autoimmune-based entity and the hepatitis C virus is suspected of being able to trigging autoimmune reactions, it has been hypothesized that hepatitis C virus infection might predispose to CD. The aim of this study was to investigate CD-related antibodies in a large series of hepatitis C virus-infected subjects that were also tested for non-organ-specific autoantibodies (NOSA) as indirect marker of autoimmune disorders. METHODS: Two hundred and forty-four patients with chronic hepatitis C virus infection (HCV-patients) and 121 patients with HCV-negative liver disease (non-HCV-patients) underwent NOSA determination and celiac serology (firstly, anti-tissue transglutaminase antibodies, then the cases which tested positive were subsequently evaluated for the presence of antiendomysial antibodies). Serum samples from 42 of the HCV-patients who underwent interferon-alpha therapy after enrollment were tested for celiac antibodies and NOSA even after stopping treatment. Additionally, sera from 1,230 blood donors were assayed for celiac serology as healthy control population. RESULTS: Positive anti-endomysial antibodies (AEA) were found in 5/244 (2%) HCV-patients, 1/121 (0.8%) non-HCV-patients and 2/1,230 (0.16%) blood donors, with a significant difference between HCV-patients and blood donors (P = 0.02; Odds ratio 12.8; 95% Confidence Interval 2.4-66). NOSA were found in 51 HCV-patients but only one of them had positive AEA. Eight out of 42 HCV-patients treated with interferon-alpha developed NOSA under therapy and none of them had CD antibodies. CONCLUSIONS: AEA occur in 2% of HCV-patients and their presence is independent of other patterns of autoimmunity.     

Increased Plasma Cell-Free DNA Level during HTNV Infection: Correlation with Disease Severity and Virus Load

Cell-free DNA (cf-DNA) in blood represents a promising DNA damage response triggered by virus infection or trauma, tumor, etc. Hantavirus primarily causes two diseases: haemorrhagic fever with renal syndrome (HFRS) and Hantavirus cardiopulmonary syndrome (HCPS), depending on different Hantavirus species. The aim of this study was to evaluate plasma cf-DNA levels in acute phase of HFRS, and to correlate plasma cf-DNA with disease severity and plasma Hanttan virus (HTNV) load. We observed the appearance of cf-DNA in 166 plasma samples from 76 HFRS patients: the plasma cf-DNA levels peaked at the hypotensive stage of HFRS, and then decreased gradually. Until the diuretic stage, there was no significant difference in plasma cf-DNA level between patients and the healthy control. Exclusively in the febrile/hypotensive stage, the plasma cf-DNA levels of severe/critical patients were higher than those of the mild/moderate group. Moreover, the plasma cf-DNA value in the early stage of HFRS was correlated with HTNV load and disease severity. In most of the patients, plasma cf-DNA displayed a low-molecular weight appearance, corresponding to the size of apoptotic DNA. In conclusion, the plasma cf-DNA levels were dynamically elevated during HFRS, and correlated with disease severity, which suggests that plasma cf-DNA may be a potential biomarker for the pathogenesis and prognosis of HFRS.